Prognosis Biomarker for Evaluating the Cure Level of Stroke Patient and a Method thereof

ABSTRACT

A method for evaluating the cure level of a stroke patient comprises following steps: (1) obtaining isolated blood sample from said stroke patient; (2) determining the concentration of serum granulocyte colony-stimulating factor (G-CSF) of said blood sample; (3) comparing the relationship between said concentration of granulocyte colony-stimulating factor (G-CSF) and the stroke severity ranking of said stroke patient; wherein United State National Institute of Health Stroke Scale (NIHSS) or modified Ranking Scale (mRS) is used in said stroke severity ranking; and (4) Using said concentration of granulocyte colony-stimulating factor (G-CSF) to predict the possible cure level of said stroke patient. The invention further provides a prognosis biomarker for evaluating the cure level of a stroke patient, and a kit containing said prognosis biomarker.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a prognosis biomarker for evaluating the curelevel of a stroke patient and a method thereof, and in particular, toendogenous granulocyte colony-stimulating factor (G-CSF) as a prognosisbiomarker for evaluating the cure level of a stroke patient, and to amethod for evaluating the cure level of a stroke patient by using thesame.

2. Description of the Prior Art

The main cause of stroke is occlusion of blood vessel at various partsin the cerebrum, causing various degree of nerve function disorder suchas abrupt deviation or paralysis of the body, blocked language, handsand feet not nimble or hand numbness, feet numbness and the like. Atpresent, clinical therapy relies mostly on drug therapy such asadministrating anti-coagulant, anti-platelet aggregating agent, and eventhrombolytic agent. In these cases, a period of time after acute stagetreatment might be needed to know whether the treatment is correct. Inaddition to common evaluating index such as reflex action, Glasgow comascale (GCS), and the like, no other biomarker can be used to evaluatethe prognosis in a short period. Mostly, the prognosis is poor, andplaces pressure on the relatives of the patient and can cost massivesocial resources as well. Prognosis is a medical term that a doctor usesto predict the disease progress of a patient, possible cure level(recovery extent), and to know whether the patient can be fullyrecovered or not.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growthfactor, and is a glycoprotein synthesized in vascular endothelial cell,monocyte and fibroblast. The accession number of granulocytecolony-stimulating factor (G-CSF) in NCBI is REGION: 35425214 . . .35427592 of NC_(—)000017(http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NC_(—)000017.9&from=35425214&to =35427592&dopt=gb), and has a sequence as depicted inSEQ ID No: 1. Said gene (G-CSF) can be transcribed to three differentmRNA variants that has accession numbers in NCBI as NM_(—)172220.1,NM_(—)000759.2, and NM_(—)172219.1, respectively.

Known functions of granulocyte colony-stimulating factor (G-CSF) can bedescribed as followed:

-   -   1. Driving hematopoietic stem cell from rest stage to        multiplication stage, and promoting the multiplication,        maturation and release of neutrophilic granulocyte;    -   2. Enhancing functions of granulocyte (phagocytosis,        chemotaxis), while giving little influence on other cells.

Recent clinical applications of granulocyte colony-stimulating factor(G-CSF) are mostly focused on disease study such as tumor chemotherapy,bone marrow graft, bone marrow suppression, AIDS, nosohemia, granulocytedeficiency, and the like; increasing leukocyte; and promoting infectionresistance.

Subcutaneous injection of exogenous granulocyte colonystimulating-factor (G-CSF) to an occluded middle cerebral artery inrecent rodent models of ischemic stroke both reduced infarction volumein the hyperacute stage (the neuroprotection effect) and enhancedfunctional recovery in subsequent subacute stages (the neurogenesiseffect) (reference 1-5).

Human in vivo studies suggest that acute ischemic stroke and acutemyocardial infarction (AMI) actually stimulate the endogenous release ofG-CSF with serum levels peaking on day 2 and remain elevated for atleast 6 days after the event (reference 6). In a small randomizedcontrol clinical trial, Shyu and colleagues demonstrated that theexogenous use of G-CSF within the first week following an ischemicstroke lead to a better prognosis and functional outcome than those inthe placebo group (reference 7). Upon review of the literature, itbecame apparent that the relationship between endogenous G-CSF secretionand the prognosis and severity of ischemic stroke had not been reportedbefore.

However, the inventor found that endogenous G-CSF might give positiveinfluence on the stem cell during acute ischemic stroke. In addition,secretion of granulocyte colony-stimulating factor (G-CSF) is involvedin the progress of human acute ischemic stroke. Accordingly, the aim ofthe invention is to investigate the relationship between granulocytecolony-stimulating factor (G-CSF) and the condition of an ischemicstroke patient, as well as to develop the prognosis biomarker forevaluating the cure level of stroke and a method thereof.

Thus, it can be seen that the above-described conventional methods forevaluating cure level of a stroke patient have many deficiencies, arenot perfect designs and need to be improved urgently.

In view of many disadvantages derived from the foregoing conventionalmethods for evaluating the cure level of a stroke patient, the inventorhad devoted to improve and innovate, and finally, has developedsuccessfully a prognosis biomarker for evaluating the cure level ofstroke patient and a method thereof according to the invention.

SUMMARY OF THE INVENTION

One object of the invention is to provide a method for evaluating thecure level of a stroke patient, characterized in that the method usesserum endogenous G-CSF of a stroke patient as the prognosis index.

Another object of the invention is to provide the prognosis biomarkerfor evaluating the cure level of a stroke patient, and a kit containingsaid prognosis biomarker for evaluating the cure level of a strokepatient.

The method for evaluating the cure level of a stroke patient that canachieve the above-mentioned objects according to the invention comprisesfollowing steps:

-   -   step 1: obtaining an isolated blood sample from said ischemic        stroke patient;    -   step 2: determining the concentration of serum granulocyte        colony stimulating-factor (G-CSF, SEQ ID No: 1) in said isolated        blood sample;    -   step 3 prognosis: when said ischemic stroke patient is evaluated        at initial stage as patient with serious symptom, and if lower        concentration of said granulocyte colony-stimulating factor        (G-CSF) in the isolated blood sample of said patient with        serious symptom is determined, it can be expected that better        cure level of said patient can be obtained after treatment, and        this indicates better prognosis; on the contrary, when said        ischemic stroke patient is evaluated at initial stage as a        patient with serious symptom, and if higher concentration of        said granulocyte colony-stimulating factor (G-CSF) in the        isolated blood sample of said patient with serious symptom is        determined, it can be expected that worse cure level of said        patient may be obtained after treatment, and this indicates        worse prognosis;

wherein said ischemic stroke patient is evaluated at initial stage as apatient with serious symptom in step 3, said evaluation method canevaluate the severity level of the symptom using suitable rankingmethod.

In a preferred embodiment of a method for evaluating the cure level of astroke patient that can achieve the above-mentioned objects of theinvention comprises following steps:

-   -   step 1: obtaining isolated sample (blood sample) from said        ischemic stroke patient;    -   step 2: determining the concentration of serum granulocyte        colony-stimulating factor (G-CSF) of said isolated blood sample;    -   step 3: at first, the relationship between the stroke severity        of said ischemic stroke patient and the concentration of        granulocyte colony-stimulating factor (G-CSF) is assessed by        means of a stroke severity ranking scale, and classify said        stroke severity ranking scores into low, middle, and        middle-high; when said stroke severity ranking scores are middle        and middle-high, it can be found that higher concentration of        granulocyte colony-stimulating factor (G-CSF) in the isolated        sample is obtained in step 2; on the contrary, when said stroke        severity ranking score is low, lower concentration of        granulocyte colony-stimulating factor (G-CSF) in the isolated        sample is obtained;    -   step 4 prognosis: said ischemic stroke patient is treated after        a suitable recovery period, and the stroke severity (cure level)        of said ischemic stroke patient is assessed by means of a stroke        severity ranking scale after said treatment, compared with the        ranking score of said stroke patient obtained in step 3 to        determine the cure level (improving level) of said patient, and        then compared with the concentration of granulocyte        colony-stimulating factor (G-CSF) determined in step 2; when the        initial ranking score of said ischemic stroke patient is middle,        and middle-high, this indicates more severe at initial stage,        and if lower concentration of said granulocyte        colony-stimulating factor (G-CSF) in the isolated blood sample        of said middle and middle-high patient is determined, it can be        expected that better cure level of said patient is obtained, and        this indicates better prognosis; on the contrary, when the        initial ranking score of said ischemic stroke patient is middle        and middle-high, this indicate the initial stage is more severe,        and if higher concentration of said granulocyte        colony-stimulating factor (G-CSF) is determined in the isolated        blood sample of said middle and middle-high patients, it can be        expected that worse cure level of said patient can be obtained,        and this indicates worse prognosis;

wherein said stroke severity ranking described in said step 3 is theUnited State National Institute of Health Stroke Scale (NIHSS);

wherein said stroke severity ranking score of less than or equal to 4points (≦4) is classified as low, 5˜7 points as middle, and higher thanor equal to 8 points (≧8) as middle-high.

In another preferred embodiment, the stroke severity ranking describedin said step 3 is modified Ranking Scale (mRS); in said modified strokeseverity ranking, classifies 0˜2 points as low, 3 points as middle, and4˜6 points as high.

Wherein the approach for ranking stroke severity described in step 3 isonly a preferred illustration, not to limit the implementation and scopeof the invention, and other suitable ranking approach may be usedinstead. Therefore, according to the foregoing, when a ischemic strokepatient is diagnosed as middle or middle-high patient at the initialstage, as lower concentration of granulocyte colony-stimulating factor(G-CSF) is determined in the isolated blood sample, it can be expectedthat a better cure level of the patient can be obtained, and thisindicates better prognosis; on the contrary, when a ischemic strokepatient is diagnosed as a middle or middle-high patient at initialstage, and higher concentration of granulocyte colony-stimulating factor(G-CSF) is determined in the isolated blood sample, it can be expectedthat a worse cure level of said patient may be obtained, and thisindicates worse prognosis.

Accordingly, the prognosis biomarker and the method for evaluating curelevel of a stroke patient in using the concentration of granulocytecolony-stimulating factor (G-CSF) as said biomarker in the isolatedblood sample of the stroke patient, and then using said concentration ofgranulocyte colony-stimulating factor (G-CSF) to predict the possiblecure level of the patient; wherein said concentration of granulocytecolony-stimulating factor (G-CSF) is in inverse proportion to the curelevel of a stroke patient.

The invention provides further a kit containing the prognosis biomarkerfor evaluating the cure level of a stroke patient. Wherein the kitcomprising suitable agents to practice the method according to thisinvention.

The invention will be illustrated by way of the following examples, butthe invention is not limited by the examples described below.

These features and advantages of the present invention will be fullyunderstood and appreciated from the following detailed description ofthe accompanying Drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of a preferred embodiment accordingto the invention and the accompanying drawings thereof will be referredin order to understand further effects of its technical content andobjects; wherein:

FIG. 1 shows the percentage of NIHSS improvement on patients havingstroke for three months, wherein concentrations of granulocytecolony-stimulating factor (G-CSF) in each group are divided into threegroups of low, middle and high concentrations; and

FIG. 2 shows the percentage of mRS on patients having stroke for threemonths, wherein concentrations of granulocyte colony-stimulating factor(G-CSF) in each group are divided into three groups of low, middle andhigh concentrations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Example 1 Material andMethod 1. Patients Selection

Total of 75 patients were recruited from the Kuang-Tien GeneralHospital, Taichung, Taiwan. Patients were included if 1) the clinicaldiagnosis was acute ischemic stroke, 2) onset was within 24 hrs, 3) thediagnosis was confirmed on brain CT or MRI and 4) they are older than 18years. Subjects were excluded if 1) the diagnosis was intracranialhemorrhage, or 2) there were other vascular abnormalities.

2. Stroke Severity Evaluation Tools

The National Institute of Health Stroke Scale (NIHSS) and modifiedRanking Scale (mRS) were used as stroke severity evaluation tools.

(1) National Institute of Health Stroke Scale, NIHSS:

The United State National Institute of Health Stroke Scale (NIHSS) isused to evaluate clinically at early stage the deficit degree of neurofunction of a patient, and even assesses the prognosis, disease severityof a patient. NIHSS is consisted of 15 items, including: level ofconsciousness (LOC), LOC questions, LOC commands, best gaze, visualfields, facial palsy, left motor arm, right motor arm, left motor leg,right motor leg, ataxia, sensory, best language, dysarthria,extinction/inattention. Its ranking score ranges from 0 to 42, thehigher the score, the more severe neurological deficit. The United StateNational Institute of Health Stroke Scale (NIHSS) assessment isperformed by neurologist having international certification of theUnited State National Institute of Health Stroke Scale (NIHSS). A timeperiod of about 5-8 minutes may be required for one complete assessment.It can assess quickly and effectively the neurological deficit degree ofa stroke patient. Its ranking score ranges as followed:

-   -   less than or equal to 4 points (≦4) is low;    -   5˜7 points is middle;    -   higher than or equal to 8 points (≧8) is middle-high.

(2) Modified Ranking Scale, mRS:

-   -   0=normal    -   1=no significant disability other than symptom; various daily        activities and affairs can be performed    -   2=low disability; unable to perform all activities, but can        administer affairs voluntarily without assist    -   3=middle disability; need some assist, but able to walk        voluntarily without assist    -   4=middle high disability; able to walk only under assist, and        unable to consider own demand without assist    -   5=high disability; clinically, incontinence and need        consistently nursing attendance    -   higher than and equal to 6=death

(3) TOTAS:

-   -   0=normal    -   1=small vessel    -   2=large atheromatous    -   3=cardioembolic    -   4=other cause    -   5=unspecific

Baseline evaluation was evaluated for all patients at admission, andblood samples were collected for analyzing granulocytecolony-stimulating factor (G-CSF) and other inflammatory marker,including: intercellular adhesion molecule-1 (sICAM-1), vascular celladhesion molecule-1 (sVCAM-1), sE-selectin; other biochemical marker,fibrinogen and highly sensitive C-reactive protein (hs-CRP), bloodlipids (including cholesterol, low density lipoprotein cholesterol, highdensity lipoprotein cholesterol, triglyceride), urea nitrogen,creatinine, total white blood cells, red blood cell, and platelet. Bloodsamples were collected from patients on one or two days after stroke tocollect data of above-mentioned markers, and conditions of said patientswere followed up continuously up to one year. In addition to assessingstroke severity of said patients by means of stroke severity ranking onDay 1-2, cure levels of 3-month recovery or 12-month recovery wereevaluated on the third month and the twelfth month, respectively,thereby the relationship between granulocyte colony-stimulating factor(G-CSF) and possible cure level or prognosis could be analyzed.

3. Assays for Intercellular Adhesion Molecule-1(sICAM-1), Vascular CellAdhesion Molecule-1(sVCAM-1) and sE-Selectin

We assayed the serum concentrations of ICAM-1, VCAM-1 and E-selectin byusing commercially available enzyme-linked immunosorbent assay kits (R&DSystems, Minneapolis, Minn., USA) in accordance with the manufacturer'sinstructions.

4. Assays for Serum G-CSF, Fibrinogen and hs-CRP

An ELISA assay kit (R&D Systems) was used for measuring serum G-CSFlevels on a 96 well microliter plate. Fibrinogen was measured by theSysmex CA6000 coagulation analyzer with Dade Behring thrombin reagent(Dade Behring, Milton Keynes, UK). Intra-assay coefficients of variationwere <4%. Highly sensitive C-reactive protein (hsCRP) was measured withBN Prospec (Dade Behring). Inter-assay and intra-assay coefficients ofvariation were <4% and <2% respectively, with a detection limit of 0.20mg/L.

5. Statistic Assay

Data were analyzed using statistical method. A Statistical Package forthe Social Sciences (SPSS; 10.5 version; Chicago, Ill., USA) was used toperform the analysis of relationship between common clinical toolmarkers and granulocyte colony-stimulating factor (G-CSF) and otherbiochemical markers.

Example 2 Result

Tertile function of SPSS was used to divide 75 acute stroke patientsinto three groups based on NIHSS/mRS scores of each patient as followed:

-   -   low NIHSS group: NIHSS≦4 points; or mRS≦2 points;    -   middle NIHSS group: 7 points≧NIHSS≧5 points; or mRS=3 points;    -   middle-high NIHSS group: NIHSS≧8 points; or 4 points≦mRS≦6        points.

Within these three groups, there was no significant variation inbaseline demographic data (see Table 1) including age, sex, body massindex, smoking index, and incidence of valvular heart disease,hypertension, hyperlipidemia and diabetes mellitus. The middle-highNIHSS group had significantly higher mRS scores (p=0.031) than those inthe low NIHSS groups. Accordingly, there was a positive relationshipbetween modified Ranking Scale (mRS) score and the United State NationalInstitute of Health Stroke Scale (NIHSS) score, and could be used asclassification criteria for evaluating stroke severity.

TABLE 1 Baseline Demographics of Subjects Grouped According to NIHSSscore Low Middle Middle-High NIHSS score ≦4 5~7 ≧8 Patient's number n =23 n = 28 n = 24 Age ± SD 69 ± 13 67 ± 50 66 ± 17 Sex (Male:Female)12:11 16:12 10:14 Body Mass Index ± SD 24 ± 4  24 ± 3  24 ± 3  SmokingIndex ± SD 17 ± 4  15 ± 4  16 ± 5  Vascular Heart disease 19% 17% 29%Hyperlipidemia 10% 13% 14% Hypertension 89% 91% 83% Diabetes Mellitus18% 30% 29% Severity of mRS score ± SD 1.8 ± 0.5  2.6 ± 0.6*  3.7 ±1.0** *p < 0.05 as compared with low NIHSS group. **p < 0.01 as comparedwith low NIHSS group. mRS: modified Ranking Scale NIHSS: NationalInstitute of Health Stroke Scale SD: standard deviation

Biochemical data from each NIHSS group (low, middle and middle-highgroups) is summarized in table II. There is no significant difference inBUN, creatinine, total cholesterol, HDL-cholesterol, LDL-cholesterol,triglyceride, sE-selectin, sICAM-1, sVCAM-1, hs-CRP, total WBC,platelets or hemoglobin across the three groups. However, concentrationsof fasting glucose, fibrinogen and G-CSF were significantly increased inthe middle-high NIHSS group when compared with the other two NIHSSgroups (p=0.023, p=0.016 and p=0.007, respectively). These indicatedthat, in severe stroke patients, concentrations of fasting glucose,fibrinogen and granulocyte colony-stimulating factor (G-CSF) were higherthan those in patients in low and middle groups.

TABLE 2 Biochemical Data Grouped According to NIHSS scores Low MiddleMiddle-High NIHSS score ≦4 5~7 ≧8 Patient's number n = 23 n = 28 n = 24BUN (mg/dL) 21.50 ± 12.33 18.74 ± 8.79 17.20 ± 5.69  Creatinine (mg/dL)1.38 ± 0.83  1.38 ± 1.10 1.08 ± 0.29 Total Cholesterol (mg/dL) 164.64 ±32.61  187.61 ± 42.94 176.81 ± 36.88  Triglyceride (mg/dL) 146.27 ±93.00  163.32 ± 89.97 199.33 ± 92.10  HDL-C (mg/dL) 44.03 ± 0.98   45.47± 11.60 39.67 ± 7.88  LDL-C (mg/dL) 91.36 ± 27.86 108.52 ± 44.45 97.28 ±41.28 E-seletin (mg/dL) 15.24 ± 13.88 13.61 ± 7.17 17.13 ± 9.52  Acsugar (mg/dL) 132.89 ± 115.35 130.60 ± 69.99  167.00 ± 112.38* ICAM-1(mg/dL) 344.59 ± 175.72  332.91 ± 172.71 386.27 ± 168.63 VCAM-1 (mg/dL)548.88 ± 181.59  560.73 ± 278.64 586.18 ± 143.02 Platelet (x1000/cmm)209.41 ± 60.32  215.71 ± 82.48 235.62 ± 94.33  RBC (x10000/cmm) 4.25 ±0.57  4.30 ± 0.51 4.92 ± 0.61 WBC (x1000/cmm) 7.24 ± 2.58  7.21 ± 2.268.20 ± 2.75 Fibrinogen (mg/dL) 378.52 ± 121.37 353.96 ± 77.73 414.42 ±99.34* hs-CRP (mg/dL) 0.53 ± 0.69  0.43 ± 0.46 0.49 ± 0.43 G-CSF (μg/L)22.07 ± 8.32  22.58 ± 8.99  30.08 ± 13.39** *p < 0.05: as compared withlow NIHSS group and middle NIHSS group. **p < 0.01: as compared with lowNIHSS group and middle NIHSS group. hs-CRP: hypersensitive C-reactiveprotein G-CSF: granulocyte colony stimulating-factor sICAM-1:intercellular adhesion molecule-1 sVCAM-1: vascular cell adhesionmolecule-1

Moreover, Table III shows the correlation analysis between NIHSS/mRSscores and selected biomarkers. As can be seen from Table 3,concentration of serum granulocyte colony-stimulating factor (G-CSF) hasa strong correlation with modified Ranking Scale (mRS) scores and theUnited State National Institute of Health Stroke Scale (NIHSS) scores onthe first day, as well as on third month and the twelfth month afterstroke, indicating that the concentration of granulocytecolony-stimulating factor (G-CSF) on 1˜2 days after stroke could be usedto evaluate the cure level on the third and twelfth months after stroke.Comparing with other biomarkers, serum G-CSF of patient is the bestbio-predictor/biomarkers of stroke severity. The P value is between1.5×10⁻⁴ to 4.4×10⁻⁵ and the Pearson correlation coefficient is between0.350 and 0.489 (R²: 12.3˜23.9).

Further, absolute improvement values (representing stroke cure level) ofthe United State National Institute of Health Stroke Scale score(ΔNIHHS) and modified ranking stroke score (ΔmRS) of stroke patientsthat have being hospitalized about three months (as shown in Table 4);while their relative improvement values (representing stroke cure level)were calculated as shown in Table 5. Data were analyzed using multipleregression model, it could be seen that, among all biomarkers, theconcentration of granulocyte colony-stimulating factor (G-CSF) was ininverse proportion to absolute improvement value (Table 4); on the otherhand, the concentration of granulocyte colony-stimulating factor (G-CSF)was in inverse proportion to relative improvement value (Table 5) andhad statistically significant difference (p<0.051. Accordingly, theconcentration of granulocyte colony-stimulating factor (G-CSF) can beused to predict the possible cure level of a stroke patient.

TABLE 3 The power of each biomarker as a predictor of NIHSS and mRSscore status. NIHSS [1-2 d] mRS [1-2 d] NIHSS [3 m] mRS [3 m] NIHSS [12m] mRS [12 m] PCC/P PCC/P PCC/P PCC/P PCC/P PCC/P WBC 0.123/0.3140.087/0.485 0.150/0.230 0.186/0.135 0.110/0.380 0.187/0.133 Ac sugar0.031/0.807 −0.068/0.601  0.067/0.607 0.066/0.609 0.026/0.8420.101/0.435 Platelet 0.133/0.270 0.176/0.155 0.139/0.261 0.222/0.0710.097/0.441 0.136/0.278 Fibrinogen 0.188/0.130 0.147/0.245 0.184/0.1450.112/0.377 0.228/0.07  0.127/0.319 hs-CRP −0.133/0.289  −0.067/0.606 −0.098/0.450  −0.152/0.238  −0.044/0.737  0.127/0.319 sICAM-1−0.006/0.958  0.001/0.998 0.061/0.611 −0.029/0.807  0.037/0.7620.020/0.871 sVCAM-1  0.02/0.869 0.056/0.653 0.052/0.680 0.071/0.5690.097/0.441 0.136/0.278 sE-seletin 0.015/0.906 0.047/0.710 0.076/0.545−0.008/0.948  0.012/0.923 0.037/0.771 G-CSF 0.456/4.4 × 10⁻⁵* 0.350/2.8× 10⁻³* 0.489/1.5 × 10⁻⁵* 0.421/2.5 × 10⁻⁴* 0.434/1.5 × 10⁻⁴* 0.395/6.4× 10⁻⁴* mRS [m]: modified Ranking Scale [month] NIHSS [m]: NationalInstitute of Health Stroke Scale[month] PCC: Pearson correlationcoefficient P: P value *Pearson correlation coefficient showedsignificantly positive correlation and P < 0.05. hs-CRP: hypersensitiveC-reactive protein G-CSF: granulocyte colony stimulating-factor sICAM-1:intercellular adhesion molecule-1 sVCAM-1: vascular cell adhesionmolecule-1 1-2 d: on 1 to 2 days stroke patients hospitalized; 3 m: onthe third month stroke patients hospitalized; 12 m: on the twelfth monthstroke patients hospitalized.

TABLE 4 The regression coefficient (β) and P value in the multivariatelinear regression analysis of absolute improvement in NIHSS and mRSscores (ΔNIHSS/ΔmRS) ΔNIHSS as the ΔmRS as the dependent dependentvariable variable β P β P G-CSF −0.453 0.008 −0.291 0.032 fibrinogen0.0113 0.440 0.0160 0.220 Ac sugar −0.135 0.318 −0.170 0.157 age −0.1650.254 −0.151 0.243 sex −0.042 0.742 −0.010 0.933 TOAST −0.188 0.180−0.190 0.131 NIHSS (1^(st) or 2^(nd) day 0.115 0.492 −0.302 0.031 afterstroke) ΔNIHSS: NIHSS scores of the first day − NIHSS scores of thethird month ΔmRS: mRS scores of the first day − mRS scores of the thirdmonth P: P value G-CSF: granulocyte colony stimulating-factor TOAST:Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification ofstroke subtype

TABLE 5 The regression coefficient (β) and P value in the multivariatelinear regression analysis of relative improvement in NIHSS and mRSscores (Δ^(R)NIHSS/Δ^(R)mRS) Δ^(R)NIHSS as the Δ^(R)mRS as the dependentdependent variable variable β P β P G-CSF −0.453 0.008 −0.335 0.032fibrinogen −0.135 0.318 −0.185 0.146 Ac sugar 0.113 0.440 0.157 0.251age −0.165 0.254 −0.184 0.173 sex −0.042 0.742 0.018 0.883 TOAST −0.1880.180 −0.260 0.049 NIHSS (1^(st) or 2^(nd) day 0.115 0.492 −0.105 0.499after stroke) Δ^(R)NIHSS: [(NIHSS scores of the first day − NIHSS scoresof the third month)/NIHSS scores of the first day] × 100% Δ^(R)mRS:[(mRS scores of the first day − mRS scores of the third month)/mRSscores of the first day] × 100% NIHSS: National Institute of HealthStroke Scale P: P value β: the regression coefficient G-CSF: granulocytecolony stimulating-factor TOAST: Trial of ORG 10172 in Acute StrokeTreatment (TOAST) classification of stroke subtype

Stroke patients (at the stroke day 1˜2) in low, middle, and middle-highgroups of the United State National Institute of Health Stroke Scale(NIHSS) were divided into three subgroups based on the concentrations ofserum granulocyte colony-stimulating factor (G-CSF) of their isolatedblood sample:

-   -   lower concentration subgroup: G-CSF<18.4 μg/L;    -   middle concentration subgroup: 18.4 μg/L≦G-CSF≦27.0 μg/L;    -   middle-high concentration subgroup: G-CSF>27.0 μg/L.

As showed in FIG. 1, three months after the stroke, the percentageimprovement in NIHSS score was good in all three G-CSF subgroups of thelow NIHSS score group, with scores of 66%, 74% and 60% respectively butno significant difference between them (p>0.05). In the middle NIHSSscore group, there was also no significant difference in the percentagechange between the G-CSF subgroups (42%, 42% and 40%, respectively;p>0.05). In the middle-high NIHSS score group however, the lower G-CSFsubgroup had greater improvements in neurologic deficits compared withthe middle and higher G-CSF subgroups (39% versus 13% and 11%,respectively; p<0.05). Obviously, in the middle-high group, comparedwith those two subgroups of middle, and high concentrations, thesubgroup of lower granulocyte colony-stimulating factor (G-CSF)concentration demonstrated more improvement of stroke cure level withrespect of neurological deficit. These indicates that, when lowerconcentration of granulocyte colony-stimulating factor (G-CSF) isdetected in the middle-high stroke patient, better cure level and hencebetter prognosis can be expected.

When using the mRS score instead, a similar result was seen. FIG. 2shows that the percentage improvement at 3 months after the stroke wasno different across the three G-CSF subgroups in the lower and middleNIHSS groups (lower NIHSS group: 50%, 36% and 57%, p>0.05; middle NIHSSgroup: 33% 41% and 22%, p>0.05 respectively). Similarly, in themiddle-high mRS group, the lower concentration G-CSF subgroups showedgreater improvement in neurologic deficit and mRS scores (50% versus 23%and 3% respectively, p<0.05). This indicates that, when lowerconcentration of granulocyte colony-stimulating factor (G-CSF) isdetected in a middle-high stroke patient, better cure level and hencebetter prognosis can be expected.

These data shows that, according to the NIHSS and mRS scoring systems,the concentration of endogenous G-CSF can be used to predict thepossible cure level of a patient 3 months after the stroke. In middleand middle-high groups according to the NIHSS and mRS scoring systems,high concentration of serum granulocyte colony-stimulating factor(G-CSF) subgroup correlates apparently with the lower percentageimprovement of the NIHSS and mRS, that is, in the middle-high strokepatient, if a lower concentration of granulocyte colony-stimulatingfactor (G-CSF) is detected, it can be expected that said middle-highstroke patient may have better cure level (improvement level)thereafter, and hence better prognosis; on the contrary, in the middleand middle-high groups of the NIHSS and mRS scores, if a higherconcentration of granulocyte colony-stimulating factor (G-CSF) isdetected in the middle-high stroke patient, a worse cure level(improvement level) and hence worse prognosis may be expected in themiddle-high stroke patient.

Besides, the present invention also measured various inflammatorymarkers to assess for a relationship with the severity of acute ischemicstroke. Although no significant relationship was seen, an increasingtrend in sICAM-1 and sVCAM-1 was noted in the middle-high NIHSS scoregroups. However, increases in fasting blood sugar, serum fibrinogen andG-CSF correlated significantly with the severity of acute ischemicstroke. Therefore, present invention provides that serum levels ofendogenous G-CSF are a better and more sensitive predictive biomarker ofseverity in acute ischemic stroke than hs-CRP, sE-selection, sICAM-1 andsVCAM-1. Analyzing the serum G-CSF concentration of patient, it can beused to validate the prognosis of acute ischemic stroke.

The prognosis biomarker and the method for evaluating the cure level ofa stroke patient provided according to the invention have followingadvantage over other conventional techniques:

By way of statistical analysis of data, the invention can providereference markers more rapidly and more accurately for a physician toevaluate the recovery status after stroke treatment, thereby not onlycan assist the evaluation of neurological regulation condition after thestroke, but also can predict the recovery condition of the patient oneyear after the stroke.

Many changes and modifications in the above described embodiment of theinvention can, of course, be carried out without departing from thescope thereof. Accordingly, to promote the progress in science and theuseful arts, the invention is disclosed and is intended to be limitedonly by the scope of the appended claims.

1. A method for evaluating the cure level of a stroke patient,comprising following steps: step 1: obtaining isolated blood sample fromsaid ischemic stroke patient; step 2: determining the concentration ofserum granulocyte colony stimulating-factor (G-CSF) of said isolatedblood sample; step 3 prognosis: when said ischemic stroke patient isdiagnosed at initial stage as a patient with serious symptom, and iflower concentration of granulocyte colony-stimulating factor (G-CSF) isdetermined in the isolated blood sample of said patient with serioussymptom, better cure level can be expected in said patient aftertreatment, and this indicates better prognosis; on the contrary, whensaid ischemic stroke patient is diagnosed at initial stage as a patientwith serious symptom, and if higher concentration of granulocytecolony-stimulating factor (G-CSF) is determined in the isolated bloodsample, worse cure level can be expected in said patient aftertreatment, this indicates worse prognosis.
 2. A method for evaluatingthe cure level of a stroke patient as recited in claim 1, wherein saidgranulocyte colony-stimulating factor (G-CSF) has a sequence as depictedin SEQ ID No:
 1. 3. A method for evaluating the cure level of a strokepatient, comprising following steps: step 1: obtaining isolated bloodsample from said ischemic stroke patient; step 2: determining theconcentration of serum granulocyte colony-stimulating factor (G-CSF) ofsaid isolated blood sample; step 3: at first, the relationship betweenthe stroke severity of said ischemic stroke patient and theconcentration of granulocyte colony-stimulating factor (G-CSF) isassessed by means of a stroke severity ranking scale, and classify saidstroke severity ranking scores into low, middle, and middle-high; whensaid stroke severity ranking scores are middle and middle-high, it canbe found that higher concentration of granulocyte colony-stimulatingfactor (G-CSF) in the isolated sample is obtained in step 2; on thecontrary, when said stroke severity ranking score is low, lowerconcentration of granulocyte colony-stimulating factor (G-CSF) in theisolated sample is obtained; step 4 prognosis: said ischemic strokepatient is treated after a suitable recovery period, and the strokeseverity of said ischemic stroke patient is assessed by means of astroke severity ranking scale after said treatment, compared with theranking score of said stroke patient obtained in step 3 to determine thecure level of said patient, and then compared with the concentration ofgranulocyte colony-stimulating factor (G-CSF) determined in step 2; whenthe initial ranking score of said ischemic stroke patient is middle, andmiddle-high, this indicates more severe at initial stage, and if lowerconcentration of said granulocyte colony-stimulating factor (G-CSF) inthe isolated blood sample of said middle and middle-high patient isdetermined, it can be expected that better cure level of said patient isobtained, and this indicates better prognosis; on the contrary, when theinitial ranking score of said ischemic stroke patient is middle andmiddle-high, this indicate the initial stage is more severe, and ifhigher concentration of said granulocyte colony-stimulating factor(G-CSF) is determined in the isolated blood sample of said middle andmiddle-high patients, it can be expected that worse cure level of saidpatient can be obtained, and this indicates worse prognosis.
 4. A methodfor evaluating the cure level of a stroke patient as recited in claim 3,where said stroke severity ranking in said step 3 and step 4 is theUnited State National Institute of Health Stroke Scale (NIHSS).
 5. Amethod for evaluating the cure level of a stroke patient as recited inclaim 4, wherein said stroke severity ranking score classifies less thanor equal to 4 points as low, 5˜7 points as middle, and higher than orequal to 8 points as middle-high.
 6. A method for evaluating the curelevel of a stroke patient as recited in claim 3, wherein said strokeseverity ranking in said step 3 and step 4 is a modified Ranking Scale(mRS).
 7. A method for evaluating the cure level of a stroke patient asrecited in claim 6, wherein said stroke severity ranking scoreclassifies 0˜2 points as low, 3 points as middle, and 4˜6 points asmiddle-high.
 8. A method for evaluating the cure level of a strokepatient as recited in claim 3, wherein said granulocytecolony-stimulating factor (G-CSF) has a sequence as depicted in SEQ IDNo:1.
 9. A prognosis biomarker for evaluating the cure level of a strokepatient, characterized in that the concentration of serum granulocytecolony-stimulating factor (G-CSF) of isolated blood sample of anischemic stroke patient is used as said prognosis biomarker.
 10. Aprognosis biomarker for evaluating the cure level of a stroke patient asrecited in claim 9, wherein said granulocyte colony-stimulating factor(G-CSF) has a sequence as depicted in SEQ ID No:
 1. 11. A prognosisbiomarker for evaluating the cure level of a stroke patient as recitedin claim 9, wherein said concentration of granulocyte colony-stimulatingfactor (G-CSF) is in inverse proportion to the possible curelevel/prognosis of said ischemic stroke patient.
 12. A prognosisbiomarker for evaluating the cure level of a stroke patient as recitedin claim 9, wherein said prognosis biomarker can further be prepared asa kit for evaluating the cure level of a stroke patient.